Recent studies have demonstrated that the monoclonal IL (interleukin)-1β-neutralizing antibody canakinumab significantly lowered rate of recurrent cardiovascular events . The NLRP3 inflammasome, an innate immune signaling complex, is the key mediator of IL-1 family cytokine production in atherosclerosis, which is activated by abundant endogenous danger signals, such as oxidized low density lipoprotein (ox-LDL) and cholesterol crystals [3, 5, 25], consistent with the concentration-dependent increase in NLRP3 inflammasome expression induced by ox-LDL in our experiment. Zhuang et al. found in vivo that KLF2-FOXP13 transcriptional signal network can inhibit the expression of endothelial inflammasome NLRP3, alleviating endothelial inflammation and delay the development of AS . In our study, we found that ox-LDL induced NLRP3 inflammasome mediated macrophages pyroptosis in a concentration-dependent manner. The expression of p-NFκB and p-IκB proteins, as markers of NF-κB signal activation, was gradually increased with the increase of ox-LDL concentration. Therefore, the above experimental results indicate that ox-LDL-induced cells pyroptosis may be associated with the activation of NF-κB signal.
Activated NF-κB plays a pivotal role in the formation and stability of atherosclerotic plaques . And in the presence of external risk factors such as high lipid induction, the key step in NF-κB activation is the phosphorylation of IκBα protein. P-IκBα is ubiquitinated and releases NF-κB dimer, which is further activated by chemical modification such as phosphorylation, and then transported to the nucleus. Liu Z et al. demonstrated that melatonin alleviates adipocyte pyroptosis by blocking NF-κB/GSDMD signaling in mouse adipose tissue,which suggests that NF-κB signaling plays a pivotal role in triggering pyroptosis. But it is not clear whether NF-κB activation has an effect on ox-LDL-induced THP-1 derived macrophage pyroptosis, which requires further study. Our experimental results indicated that inhibition of NF-κB signal activation can effectively diminish ox-LDL-induced cell pyroptosis via inhibiting the activation of NLRP3 inflammasome, suggesting that NF-κB/NLRP3 pathway was required for ox-LDL induced macrophages pyroptosis, consistent with previous researches.
A recent study has shown that NF-κB activation is associated with ABCA1 expression and cholesterol efflux in macrophages. In our study, we found that NF-κB signaling activation inhibitors significantly blocked ox-LDL inhibition of ABCA1 expression and increased cholesterol efflux in foam cells, which suggests that under ox-LDL treatment, NF-κB signal acts as the upstream of ABCA1 and is activated by ox-LDL to inhibit the expression of ABCA1, impeding the reverse cholesterol transport, and aggravating the degree of cell foaming.
However, it is not clear whether ABCA1 can regulate the pyroptosis of macrophage derived foam cells. In 2018, a study showed that cholesterol accumulation in bone marrow cells activates NLRP3 inflammasomes, further contributing to the development of AS plaques . In myeloid ABCA1/ G1-deficient LDLR -/- mice, the deletion of NLRP3 or caspase-1/11 reduced the size of atherosclerotic lesions . In our study, we demonstrated for the first time in vitro that the inhibition of cholesterol transport capacity in macrophage-derived foam cells increases the expression of NLRP3 inflammasome, thereby regulating cell pyroptosis and triggering inflammatory response.
Although some studies have shown that pyroptosis activates the inflammatory network and releases a large number of inflammatory factors, promoting the development of atherosclerosis, which is adverse to the development of AS and the stability of plaques [18, 23]. However, several studies have shown that cell pyroptosis plays an important role in controlling microbial infection. Costa Franco MMS et al. found that in the dendritic cells of C57BL/6 mice infected with Brucella, the genomic DNA of Brucella can cause pyroptosis of bone marrow-derived dendritic cells, which has been shown to help control the infection and accelerate the clearance of the pathogen . Wang et al. have recently shown that pyroptosis induced inflammation can trigger a powerful anti-tumor immune effect , which is conducive to tumor clearance. The above studies indicate that moderate pyroptosis which is an important immune defense response of the body and plays an important role in resisting infection and endogenous danger signals, can quickly remove pathogens and risk factors, enhancing the immune function. It was previously reported that treatment of smooth muscle cells with VX-765 at 50µM effectively reduced pyroptosis. Therefore, in this study, we treated macrophages with 50 µM VX-765, and found that ox-LDL-induced cell pyroptosis was significantly reduced. We also found that the accumulation of lipid droplets in foam cells was higher, and the intracellular cholesterol efflux was decreased which suggests that cell pyroptosis may be a double-edged sword in the development of AS.
In conclusion, NF-κB/ABCA1 pathway aggravation of ox-LDL-induced cell pyroptosis has been demonstrated in THP-1-derived macrophages for the first time, providing a novel therapeutic avenue for the treatment of AS.